Rotaviruses are the most important cause of severe dehydrating diarrhea in young children. The mortality due to rotavirus infection is significant, globally causing nearly 1 million deaths each year. Recently, a vaccine was developed in the Laboratory of Infectious Diseases that provides a high level of protection against severe rotaviral diarrhea. Because of the pathogenic importance, an overall goal of the Laboratory remains the development of new vaccines, the improvement of existing vaccines, and the identification of other methods for preventing and treating rotaviral disease. Accomplishing this goal would be helped by a more complete understanding of the molecular biology of the rotavirus. In particular by defining events in the replication and packaging of the viral genome, we should gain the information necessary to attenuate pathogenic strains of rotavirus and thereby to molecularly engineer new candidate vaccines. The specific focus of this project is to characterize the fundamental events in the replication of the rotavirus genome which then can be used to develop a reverse genetics system for manipulating the genetic information of the virus.The core of the rotavirus virion, which contains eleven segments of double-stranded (ds)RNA, is formed by three proteins: the viral RNA polymerase VP1, the core shell protein VP2, the capping enzyme VP3. During genome replication, viral messenger (m)RNAs are packaged into core-like replication intermediates, which then use the mRNAs as templates to synthesize dsRNA. This project will identify the location and structure of recognition signals in viral mRNAs that participate in RNA packaging and replication and will define the specificity and function of the RNA-binding proteins involved in these events. This will be accomplished by (i) performing cell-free replication assays on mutated viral mRNAs, (ii) computer modeling and structural mapping of recognition signals in mRNAs, (iii) analyzing the enzymatic and structural properties of recombinant viral proteins, and (iv) defining the specificity and targets of the viral RNA-binding proteins. An overall technical aim of this project is to incorporate its findings into the development of a cell-free system which allows the assembly of infectious particles from recombinant protein and RNA. - Rotavirus, Infantile diarrhea, RNA replication, RNA packaging, Protein structure, Protein function, RNA polymerase